Craniofacial malformations arise by errors in development, as can occur when genes that pattern the embryo do not function correctly. Learning about how the facial skeleton forms, as well as the genes that control its formation, is essential for the eventual understanding of the bases of inherited craniofacial disorders. The zebrafish provides a useful animal model for such studies: The zebrafish embryo is amenable for developmental and genetic investigation. Its embryonic head skeleton shares considerable similarity with that of humans. Genes that pattern zebrafish and human skeletal development have been highly conserved during evolution, hence understanding gained from zebrafish can apply directly to human development. This proposal is to examine critically a period of about a day of embryonic development in the zebrafish when cells form the rudiments of the facial skeleton. A "morphogenetic cascade" hypothesis proposes that, under genetic control, neural crest-derived cells in the pharyngeal arch primordia undergo a short series of rearrangements that, at each step, refine skeletal prepatterning. The first specific aim is to identify the cells, by fate mapping at successive steps of the cascade, that will form specific facial skeletal elements. The second aim is to watch the cells in the intact embryo directly, by making time-lapse recordings with a confocal microscope (4d-analysis), to learn whether and precisely how the predicted cellular rearrangements occur. Accomplishment of these two aims will reveal the cellular behaviors that build individual elements of the head skeleton. The third specific aim is to study the cascade genetically, by analyzing craniofacial mutants and patterning genes cloned by homology. Hierarchically upstream regulatory genes have already been identified by these techniques and the principal focus will be to identify and characterize new genes that execute that later, downstream, steps in the cascade.